Project summary. M. tuberculosis succeeds as a pathogen based on its ability to survive within the endosomal network of infected cells and persist in host tissues for decades. Microscopic examination of mycobacteria growing in tissues shows that they undergo a substantial transformation of their lipid-laden cell walls during growth in cells. Recent studies have identified two mycobacterial lipid synthesis systems that contribute to the remodeling process. The applicant's laboratory has discovered a new class of mycobacterial lipids produced by polyketide synthase 12 (Pks12) (Nature 404, p. 884;J Exp Med 200, p. 1559). Also, sigma factor L (sigL) regulates the expression of polyketide synthase 7 (Pks7) and polyketide synthase 10 (PkslO) (J Bact., in press). Both systems are determinative for the outcomes of M. tuberculosis infection in vivo, but the structures and functions of lipid intermediates controlled by these newly discovered systems are not known. We now propose to measure the effects of pks7 and pkslO on infection in vivo and to identify global changes in cell wall lipids during adaptation of M. tuberculosis to growth in lung tissue and myeloid cells. These studies use targeted gene deletion and global analysis of cell wall structure with a newly developed experimental system for rapid and nearly comprehensive monitoring of cell wall lipids. This system uses orthogonal liquid chromatography directly coupled to high throughput mass spectrometry with software-assisted analysis to detect the small number of altered lipids from among the thousands of molecular ions screened. The complete structures of lipids altered by these stimuli will be identified using collision-induced dissociation mass spectrometry, Fourier transform ion cyclotron mass spectrometry and nuclear magnetic resonance. These experiments combine targeted and global lipid detection to uncover systems used by M. tuberculosis to adapt to growth conditions in the host. Relevance: M. tuberculosis has infected 1.7 billion humans and kills 2 million patients annually, yet there is no simple and effective treatment for tuberculosis. Experts have argued that development of a drug or vaccine that kills all bacteria in the early phase of infection could eliminate tuberculosis in developed countries. Identification of the genes, enzymes and cell wall lipids that mediate adaptation to growth in tissues provides the information necessary to achieve this goal.